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1.
The guinea pig cytomegalovirus (GPCMV) is unique among the cytomegaloviruses of small mammals, insofar as during pregnancy it crosses the placenta, causing infection of the fetus. Although the guinea pig model is well suited to vaccine studies, the lack of cloned, recombinant forms of immunogenic GPCMV proteins, such as envelope glycoproteins, has hindered experimental evaluations of subunit immunization for prevention of fetal disease. Since the glycoprotein B (gB) is a major target of neutralizing antibody responses, the GPCMV gB was cloned and expressed in a recombinant baculovirus. A recombinant was generated which expressed gB, truncated at codon 692, upstream of the putative transmembrane domain. Processing and expression of the recombinant protein, designated Bac-gB, was assessed, and the protein was characterized immunologically. Anti-gB antibodies were immunoreactive with Bac-gB by enzyme linked immunosorbent assay (ELISA) and immunoblot assay. Immunoprecipitation with polyclonal anti-GPCMV antisera identified protein species of 120, 80 and 30 kDa by reducing SDS-PAGE, suggesting that authentic cleavage and processing of Bac-gB occurred in insect cells. Sera from guinea pigs immunized with lectin-column purified native glycoproteins had high ELISA titers to Bac-gB. Recombinant GPCMV gB expressed in insect cells should prove useful in defining correlates of protective immunity in the GPCMV congenital infection model. 相似文献
2.
Mark R Schleiss 《Journal of clinical virology》2008,41(3):224-230
Vaccines are urgently needed to protect newborns against the devastating sequelae of congenital cytomegalovirus infection. Evaluation of candidate vaccines in the guinea pig model of congenital infection can shed light on potentially useful strategies for humans, since guinea pig CMV (GPCMV) is transmitted to the fetus transplacentally, causing infection and disease in utero. A number of vaccine strategies have been evaluated in this model, including DNA vaccines, live attenuated vaccines, and recombinant glycoprotein vaccines. Induction of virus-neutralizing antibody appears to play a key role in protection of the fetus. Recently, a vectored vaccine based on the GPCMV homolog of the UL83 (pp65) protein has also been shown to be effective when used as a preconceptual vaccine in this model, suggesting that strategies designed to elicit T-cell responses may be of value in protection of the fetus. 相似文献
3.
Biotin labelled hybridization probes prepared from recombinant plasmids containing segments of the guinea pig cytomegalovirus (GPCMV) genome were used to detect GPCMV nucleic acids in guinea pig cells by in situ hybridization. The time course of GPCMV infection was assessed in two cultured cell types, guinea pig embryo (GPE) cells and 104C1 cells, a transformed and cloned guinea pig cell line. Detection of GPCMV nucleic acids was accomplished in both cell types with individual GPCMV DNA fragments and with mixtures of GPCMV DNA fragments. When compared to other established methods of GPCMV detection, the method of in situ hybridization enabled the detection of a higher percentage of positive cells early during the course of the infection. In addition, differences in the replication cycle of GPCMV in the two cultured cell lines could be demonstrated. These findings will facilitate future studies of GPCMV tissue tropism in vivo. 相似文献
4.
Junin virus infection in guinea pigs is known to be similar to human Argentine hemorrhagic fever (AHF). The guinea pig was chosen as a model for transplacental transmission of Junin virus, as both guinea pig and man have a similar placental structure. Pregnant guinea pigs were infected with the pathogenic XJ strain of Junin virus intramuscularly route at different stages of pregnancy. The group infected during the last third of pregnancy produced 16 newborn, but mortality reached 100%: 18% were born with typical AHF hemorrhagic signs, 54% without signs, and the remainder were stillborn. Virus was recovered from organs of newborns, as well as placental tissues. A second group, infected in the second third of pregnancy, died with intrauterine fetuses, all of which showed hemorrhagic signs and virus present. In a last group, infected in the first third of pregnancy, fetuses were free from macroscopic lesions. In order to determine whether lactation may be an alternative infection route in guinea pigs, mother guinea pigs were infected with Junin virus at different times postparturition. The 84% noninfected newborn housed together with their infected mothers died during the suckling period, half with typical AHF signs. Junin virus transmission from mother to fetus was thus proved, and lactation may be considered as an alternative perinatal infection route. 相似文献
5.
6.
Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: growth characteristics and antigentic relationship.
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The growth characteristics of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in cell cultures were compared. Guinea pig fibroblast cells were highly susceptible to infection with both viruses, whereas guinea pig kidney cells were sensitive only to GPHLV. No cytopathic effect was observed in the latter cell system after infection with GPCMV,nor was there an increase in virus titer, although the cirus persisted in the kidney cells for 2 to 3 weeks postinfection. Electron microscope studies showed nonvirion tubular structures in GPCMV -infected fibroblast cells, but not in GPHLV- infected cells. Large packages of enveloped nuclear virus particles were commonly seen in GPHLV -infected cells, especially kidney epithelial cells, but none were found in the GPCMV -infected fibroblasts. Complete enveloped extracellular virus particles were present in both virus-cell systems. Both viruses showed narrow host spectra and replicated well only in guinea pig cells although GPHLV multiplied to some degree in rabbit cells. No antigenic relationship could be demonstrated between the two viruses using antisera specific for each virus that was produced in rabbits and guinea pigs. Rabbits produced high neutralizing antibody titers to GPHLV, whereas guinea pigs were the animals of choice for GPCMV antiserum production. 相似文献
7.
Summary Intranuclear localization of viral antigens in guinea pig cytomegalovirus (GPCMV) infected guinea pig embryo (GPE) cells was investigated by cross-reactive indirect immunoperoxidase and immunoferritin techniques utilizing guinea pig antisera to GPCMV. Following primary fixation with 4 percent paraformal-dehyde, a brief treatment of infected cells with 0.25 percent trypsin was found to enhance penetration of antibodies and the conjugates. Ferritin or horseradish peroxidase conjugated goat anti-rabbit IgG was used as a secondary antibody that cross reacted with guinea pig immunoglobulins in order to reduce non-specific immunochemical reactions. Using light microscopy following immunoperoxidase staining, GPCMV antigens in an intranuclear location were not discernable when the infected cells were stained without pretreatment with trypsin, however intranuclear GPCMV antigens could be visualized after the fixed cells were treated with trypsin for 2–4 minutes prior to addition of the antiserum. Electron microscopic examination following indirect immunoferritin staining revealed viral antigens localized on viral capsids and on scattered electrondense amorphous matrices but not on the surrounding tubular structures or fibrils. The possibility that tubular structures may be a host cell product produced in response to GPCMV infection is discussed.With 3 Figures 相似文献
8.
Schleiss MR Bourne N Bravo FJ Jensen NJ Bernstein DI 《Journal of virological methods》2003,108(1):103-110
Human cytomegalovirus (HCMV) is the most common cause of congenital viral infection in the developed world, and can lead to significant morbidity. Animal models of HCMV infection are required for study of pathogenesis, because of the strict species-specificity of cytomegalovirus (CMV). Among the small animal CMV models, the guinea pig CMV (GPCMV) has unique advantages, in particular its propensity to cross the placenta, causing disease in utero. In order to develop quantitative endpoints for vaccine and antiviral therapeutic studies in the GPCMV model, a quantitative-competitive PCR (qcPCR) assay was developed, based on the GPCMV homolog of the HCMV UL83 gene, GP83. Optimal amplification of GPCMV DNA was observed using primers spanning a 248 base pair (bp) region of this gene. A 91 bp deletion of this cloned fragment was generated for use as an internal standard (IS) for PCR amplification. Standard curves based upon the fluorescent intensity of full-length external target to IS were compared with signal intensity of DNA extracted from blood and organs of experimentally infected guinea pigs in order to quantify viral load. Viral load in newborn guinea pigs infected transplacentally was determined and compared with that of pups infected with GPCMV as neonates. Viral loads were highest in pups infected as neonates. The most consistent isolation and highest quantities of viral DNA were observed in liver and spleen, although viral genome could be readily identified in brain, lung, and salivary gland. Viral load determination should be useful for monitoring outcomes following vaccine studies, as well as responses to experimental antiviral agents. 相似文献
9.
Histopathologic and ultrastructural studies of disseminated cytomegalovirus infection in strain 2 guinea pigs 总被引:2,自引:0,他引:2
C K Fong H Lucia F J Bia G D Hsiung 《Laboratory investigation; a journal of technical methods and pathology》1983,49(2):183-194
Inbred strain 2 guinea pigs developed severe disseminated disease during acute experimental guinea pig cytomegalovirus (GPCMV) infection. A high mortality rate (100%) resulted, with most animals dying between 10 and 14 days after high dose (7.5 X 10(5) TCID50) virus inoculation. Infectious virus was recovered from many tissues, including spleen, lungs, liver, pancreas, heart, adrenals, kidneys, and salivary glands. The rate of GPCMV isolation from these tissues ranged from 50 to 100%. Gross lesions were observed in the spleen, liver, and lungs. On histologic examination, lesions were also seen in many other organs, including heart, pancreas, kidneys, adrenals, brain, intestines, and salivary glands. Intranuclear viral inclusions were present in many cell types of various organs. Under electron microscopic examination, cells with viral inclusions were easily found in the spleen, and liver, but less readily in the lungs, kidneys, salivary glands, and other organs. Most of the intranuclear inclusions consisted of electron-dense fibrils (10 nm diameter), viral nucleocapsids (100 nm), and tubular structures (60 nm diameter). Dense bodies and enveloped dense virions containing single or multiple capsids were present in the cytoplasm of many infected cells. The morphologic developments of GPCMV in these visceral tissues of strain 2 guinea pigs resembled those seen in GPCMV-infected cultured guinea pig cells but differed from those observed in the infected salivary gland duct cells. Strain 2 guinea pigs are a useful animal model for studying disseminated infection in CMV-associated human diseases. 相似文献
10.
Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: pathogenesis and persistence in experimentally infected animals.
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The pathogenesis of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in guinea pigs was compared. Animals were inoculated with the two viruses by different routes and sacrificed after varying periods of time. GPCMV was consistently isolated from salivary gland 2 weeks postinoculation and thereafter following intraperitoneal or subcutaneous incoulaton. Virus was less frequently found in other tissues including blood, spleen, and kidney. Intranuclear inclusions were seen in tissue sections of salivary gland after inoculation with GPCMV- infected tissue suspension, but were only rarely found after inoculation with tissue culture virus. In GPHLV-infected guinea pigs, consistent latent infection of leukocytes and other tissues was detected by cocultivation techniques. Intranuclear inclusions were not found in the spleen, salivary gland, or other infected tissues after GPHLV infection with either tissue culture virus or infected tissue suspension. Guinea pigs inoculated with GPCMV produced high titers of specific neutralizing antibody to the homologous virus; those inoculated with GPHLV developed long-term viremia accompanied by minimal neutralizing antibody levels to the virus. 相似文献
11.
Immunogenicity evaluation of DNA vaccines that target guinea pig cytomegalovirus proteins glycoprotein B and UL83 总被引:3,自引:0,他引:3
Vaccines are needed for control of congenital human cytomegalovirus (HCMV) infection. Although the species-specificity of cytomegaloviruses precludes preclinical evaluation of HCMV vaccines in animal models, the guinea pig cytomegalovirus (GPCMV), which causes disease in utero, is a relevant model for the study of vaccines against congenital infection. We investigated whether DNA vaccines that target two GPCMV proteins, glycoprotein B (gB) and UL83 (pp65), are capable of eliciting immune responses in vivo. After cloning each gene into an expression vector, DNA was delivered by intramuscular inoculation and by pneumatic epidermal delivery. In Swiss-Webster mice, anti-gB titers were significantly higher after epidermal delivery. After epidermal inoculation in guinea pigs, all gB-immunized animals (n = 6) had antibody responses comparable to those induced by natural infection. Viral neutralization titers ranged from 1:64 to greater than 1:128. A GPCMV UL83 DNA vaccine also elicited an antibody response in all immunized guinea pigs (n = 6) after epidermal administration. Immunoprecipitation and Western blot assays confirmed that immune sera were immunoreactive with virion-associated UL83 and gB proteins. We conclude that DNA vaccines against GPCMV structural proteins are immunogenic, and warrant further investigation in the guinea pig model of congenital CMV infection. 相似文献
12.
Summary Salivary glands from Hartley guinea pigs were experimentally infected with guinea pig cytomegalovirus (GPCMV) and examined by light and electron microscopy at different time intervals. Characteristic intranuclear and intracytoplasmic viral inclusions were observed in duct cells of infected animals. Viral inclusion counts and infectivity titers in the salivary gland reached maximum levels by 3 to 4 weeks after infection; infectivity persisted, though at reduced levels, for at least 30 weeks. Electron microscopic examination of viral inclusions revealed several developmental events including nucleocapsid assembly, envelopment of nucleocapsids at the inner nuclear membrane and their enclosure by a thin vacuolar membrane. While contained within cytoplasmic vacuoles, enveloped virions acquired surface spikes. Cytoplasmic vacuoles containing virions subsequently coalesced and discharged mature virions at the cell surface into the lumen of the salivary gland duct. The data indicate that the ultrastructural development of GPCMV in the guinea pig salivary gland shows many similarities to that of human cytomegalovirus in humans. The salivary gland may provide a primary locus for virus shedding and horizontal transmission of cytomegalovirus.With 7 Figures 相似文献
13.
套式聚合酶链反应在豚鼠巨细胞病毒宫内感染模型中的应用价值 总被引:2,自引:0,他引:2
目的运用套式聚合酶链反应(N-PCR)检测豚鼠巨细胞病毒(GPCMV)宫内感染,并探讨N-PCR在模型建立中的应用价值.方法选择无感染史豚鼠,受孕后腹腔接种GPCMV,分娩后24h内处死;观察妊娠结局,运用N-PCR检测亲代及予代感染情况,并对标本进行病理学检查.N-PCR的特异性实验设阴性、空白和阳性对照.结果感染孕鼠出现病毒血症征象,胎仔出现流产、死胎等异常结局.N-PCR检测显示,亲代100%(10/10)感染,子代96.0%(24/25)感染,且感染脏器均出现非特异性组织病理学变化.N-PCR有较高的GPCMV检测特异性和灵敏性.结论N-PCR检测GPCMV宫内感染,简便、快速、灵敏、准确,是研究模型建立的一种良好方法. 相似文献
14.
Summary Subunit vaccines which target viral envelope glycoproteins offer promise for the prevention of congenital cytomegalovirus (CMV) infection. The guinea pig model of CMV infection is uniquely well suited to testing vaccines for prevention of congenital infection, since, in contrast to other animal cytomegaloviruses, the guinea pig CMV (GPCMV) crosses the placenta, producing intrauterine infection. Antibody to the CMV glycoproteins B (gB) and H (gH) appears to be important in conferring protective immunity. Unfortunately, little is known about specific GPCMV envelope glycoproteins. Sequencing of GPCMV genome fragments was therefore undertaken to test whether GPCMV encodes a gH homologue. Partial sequencing of theHind III A fragment of the GPCMV genome revealed an open reading frame of 2 169 nucleotides capable of encoding a protein of 723 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gH coding sequences of other herpesviruses. The GPCMV gH ORF encodes 12 highly conserved cysteine residues, contains 9 potential N-linked glycosylation sites, and has a predicted Mr of 81.6 kDa. Northern blot hybridizations with gH-specific probes identified an abundant 5.1 kb mRNA with expression kinetics of an early gene. A polyclonal antiserum raised against a synthetic peptide derived from the deduced amino acid sequence of the gH ORF identified a virion-associated protein with an approximate Mr of 85-kDa, the putative GPCMV gH, in immunoblot assays.The nucleotide sequences reported in this paper have been submitted to the GenBank database and assigned the accession number U49361. 相似文献
15.
Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies. 相似文献
16.
17.
Jacques Izard 《Anatomical record (Hoboken, N.J. : 2007)》1966,155(1):117-132
Thymic reticular cells of the guinea pig were studied at different stages of its development and involution: the fetus of the last third of pregnancy, the newborn, the puberal adult and the adult implanted with estradiol. Most of the reticular cells were epithelial with desmosomes and tonofilaments originating from the endodermic primordium. In the medulla, some of these epithelial cells appeared to be transformed into elements of Hassall's corpuscles with an increase of tonofilaments in close association with keratohyalin granules. Moreover, phagocytic reticular cells were occasionally seen. Their origin remains unknown (epithelial or menschymal ?). The problem of the thymic secretion is discussed. There was no convincing evidence of a secretion of protein or polypeptid inside these reticular cells whatever the age of the animal. However, it is not possible to exclude the presence of another form of endocrin secretion, especially a colloid or a steroid secretion, at least in the fetus and newborn thymus. 相似文献
18.
Gisela Enders Anja Daiminger Ursula Bäder Simone Exler Martin Enders 《Journal of clinical virology》2011,52(3):244-246
Background: The risk of intrauterine cytomegalovirus (CMV) infection and disease in the fetus or newborn largely depends on time of primary maternal infection during pregnancy.
Objectives
Prospective cohort study of pregnancy outcome in relation to gestational age at primary maternal CMV infection.Study design
In a total of 248 pregnancies with primary infection the onset of infection was determined by IgG seroconversion, IgG avidity and/or onset of clinical symptoms. Congenital infection was diagnosed by CMV detection in amniotic fluid, fetal tissue or urine of the neonate in the first 2 weeks of life. Clinical symptoms were retrieved from ultrasound and medical records.Results
The intrauterine transmission rates following primary CMV infection in the pre- and periconceptional period were 16.7% (4/24) and 34.5% (10/29), respectively. For the first, second and third trimester of pregnancy transmission rates were 30.1% (25/83), 38.2% (29/76) and 72.2% (26/36), respectively. The rate of symptomatically infected fetuses or newborns at birth was 22.8% for any symptoms and 10.3% for severe manifestations. No symptoms were observed in infected newborns of mothers with primary infection in the preconceptional period and in the third trimester.Conclusions
The risk of intrauterine transmission following primary maternal infection in the third trimester is high, but the risk of neonatal disease is low. The highest risk of severe symptoms in the fetus and newborn exists around conception and in the first trimester of pregnancy. 相似文献19.
Bacq Y 《Pathologie-biologie》1999,47(9):958-965
Transaminase level elevation during pregnancy should be viewed as abnormal and evaluated. A high index of suspicion for acute fatty liver of pregnancy should be maintained during the third trimester, since early delivery has radically transformed the maternal and fetal prognosis of this condition. Pruritus is the main symptom of intrahepatic cholestasis, which carries a risk for the fetus. Urinary tract infection can cause cholestasis or worsen intrahepatic cholestasis of pregnancy. In patients with preeclampsia, rapid delivery should be considered if there is evidence of HELLP syndrome. Patients with mild chronic viral hepatitis can usually carry a pregnancy to term without undue difficulty. Neonates born to HBsAg-positive mothers should receive HBV-Ig and vaccine at birth to prevent perinatal transmission of the HBV. In patients with chronic hepatitis C, serum transaminase levels often return to normal during pregnancy, although the virus remains detectable in the blood. Mother-to-infant transmission of the HCV is possible but fairly uncommon if the mother is HIV-negative. 相似文献
20.
We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication. 相似文献